Experimental observations have over the past few years suggested that astrocytes play a more active role in brain function than previously recognized. It is therefore of importance to establish how astrocytes communicate with other cells. In previous observations, we have reported that astrocytic calcium waves are mediated by release of nucleotides and activation of purinergic receptors in neighboring cells. Our intention in this proposal is to identify the mechanism of astrocytic ATP release, as well as to define the cellular events that result in ATP efflux. We first plan to test the hypothesis that efflux of cytosolic ATP is mediated by opening of Cx43 hemichannels. Combining single channel recordings using an inside-out configuration, together with bioluminescence imaging, will enable us to directly establish whether Cx43-hemichannels release ATP. Next we will ask whether the lack of ATP release from neurons and oligodendrocytes reflect their lack of Cx43 expression. We will evaluate the effect of adenoviral transfer of cDNA encoding Cx43 together with an EGFP reporter upon ATP release. These experiments also explore the alternative possibility that the lack of ATP bursting in neurons reflect intrinsic cellular properties, rather than lack of Cx-hemichannels by overexpression of the neuronal gap junction protein, Cx36, in glial cells. We will also ask whether oscillatory calcium increments is a necessary prerequisite and thereby apredictor of ATP burst releases. If so, this approach that will allow us to characterize the cellular events, which result in ATP release. To this end, we will combine bioluminescence detection of ATP with calcium imaging, so as to define calcium dynamic in cells that release ATP. These experimentsoffer the first concurrent visualization of a transmitter and calcium signaling in live cells. Defining the pathways by which astrocytes signal among one another, as well as to their neuronal partners, may provide us an important basis for the directed manipulation of neuronal-glial signaling, both in health and disease.